Putative Retinoic Acid Response Elements Research Articles

All-trans retinoic acid suppresses bone morphogenetic protein 4 in mouse diabetic nephropathy through a unique retinoic acid response element.

Diabetic nephropathy (DN) causes mesangial matrix expansion, which results in glomerulosclerosis and renal failure. Collagen IV (COL4) is a major component of the mesangial matrix that is positively regulated by bone morphogenetic protein 4 (BMP4)/suppressor of mothers against decapentaplegic (Smad1) signaling. Because previous studies showed that retinoids treatment had a beneficial effect on kidney disease, we investigated the therapeutic potential of retinoids in DN, focusing especially on the regulatory mechanism of BMP4. Diabetes was induced with streptozotocin in 12-wk-old male Crl:CD1(ICR) mice, and, 1 mo later, we initiated intraperitoneal injection of all-trans retinoic acid (ATRA) three times weekly. Glomerular matrix expansion, which was associated with increased BMP4, phosphorylated Smad1, and COL4 expression, worsened in diabetic mice at 24 wk of age. ATRA administration alleviated DN and downregulated BMP4, phosopho-Smad1, and COL4. In cultured mouse mesangial cells, treatment with ATRA or a retinoic acid receptor-α (RARα) agonist significantly decreased BMP4 and COL4 expression. Genomic analysis suggested two putative retinoic acid response elements (RAREs) for the mouse Bmp4 gene. Chromatin immunoprecipitation analysis and reporter assays indicated a putative RARE of the Bmp4 gene, located 11,488-11,501 bp upstream of exon 1A and bound to RARα and retinoid X receptor (RXR), which suppressed BMP4 expression after ATRA addition. ATRA suppressed BMP4 via binding of a RARα/RXR heterodimer to a unique RARE, alleviating glomerular matrix expansion in diabetic mice. These findings provide a novel regulatory mechanism for treatment of DN.

Genomic Knockout of Two Presumed Forelimb Tbx5 Enhancers Reveals They Are Nonessential for Limb Development

SUMMARYA standard approach in the identification of transcriptional enhancers is the use of transgenic animals carrying DNA elements joined to reporter genes inserted randomly in the genome. We examined elements near Tbx5, a gene required for forelimb development in humans and other vertebrates. Previous transgenic studies reported a mammalian Tbx5 fore-limb enhancer located in intron 2 containing a putative retinoic acid response element and a zebrafish tbx5a forelimb (pectoral fin) enhancer located downstream that is conserved from fish to mammals. We used CRISPR/Cas9 gene editing to knockout the endogenous elements and unexpectedly found that deletion of the intron 2 and downstream elements, either singly or together in double knockouts, resulted in no effect on forelimb development. Our findings show that reporter transgenes may not identify endogenous enhancers and that in vivo genetic loss-of-function studies are required, such as CRISPR/Cas9, which is similar in effort to production of animals carrying reporter transgenes.

Aromatase activity induction in human adipose fibroblasts by retinoic acids via retinoic acid receptor α

Estrogen synthesis in adipose tissue is associated with the development of breast cancer. Tumors are preferentially found in breast quadrants with strongest expression of the cytochrome P450 aromatase (encoded by the gene CYP19A1). Several promoters regulated by various hormonal factors drive aromatase expression in human breast adipose fibroblasts (BAFs). As adipose tissue is a major source of retinoids, in this study, we investigated their role in the regulation of aromatase expression. The retinoids all-trans-retinoic acid (at-RA) and 9-cis-RA induce aromatase activity in human BAFs. In BAFs, at-RA induces aromatase gene expression via promoter I.4. In 3T3-L1 cells, both retinoids specifically drive luciferase reporter gene expression under the control of aromatase promoter I.4, whereas other promoters active in human adipose tissue are insensitive. Activation by retinoids depends on a 467 bp fragment (-256/+211) of promoter I.4 containing four putative retinoic acid response elements (RAREs). Site-directed mutagenesis revealed that only RARE2 (+91/+105) mediates the retinoid-dependent induction of reporter gene activity. In 3T3-L1 preadipocytes and human BAFs, RA receptor α (RARα (RARA)) expression is predominant, whereas RARβ (RARB) or RARγ (RARG) expression is low. Electrophoretic mobility shift assays with nuclear extracts obtained from human BAFs and 3T3-L1 cells identified a specific RARE2-binding complex. Retinoids enhanced complex formation, whereas pre-incubation with anti-RARα antibodies prohibited the binding of RARα to RARE2. Chromatin immunoprecipitation showed RA-dependent binding of RARα to the RARE2-containing promoter region in vivo. Furthermore, we provide evidence that RARE2 is also necessary for the basal activation of promoter I.4 in these cells. Taken together, these findings indicate a novel retinoid-dependent mechanism of aromatase activity induction in adipose tissue.

Identification of a retinoic acid‐responsive neural enhancer in the Ciona intestinalis Hox1 gene

The Hox1 gene in the urochordate ascidian Ciona intestinalis (Ci-Hox1) is expressed in the nerve cord and epidermis. We identified a nerve cord enhancer in the second intron of Ci-Hox1, and demonstrated that retinoic acid (RA) plays a major role in activating this enhancer. The enhancer contained a putative retinoic acid-response element (RARE). Mutation of the RARE in the Ci-Hox1 nerve cord enhancer only partially abolished the enhancer activity. Genes encoding RA synthase and the RA receptor were knocked down using specific antisense morpholino oligos (MOs), and injection of embryos with these MOs resulted in the complete disappearance of epidermal expression of Ci-Hox1 and reduction of neural expression. However, nerve cord expression was not completely repressed. These results suggest that the nerve cord enhancer is activated by two partially redundant pathways; one RA-dependent and one RA-independent.

Hepatic Cyp2d and Cyp26a1 mRNAs and Activities Are Increased During Mouse Pregnancy

There is considerable evidence that drug disposition is altered during human pregnancy and based on probe drug studies, CYP2D6 activity increases during human pregnancy. The aim of this study was to determine whether the changes of CYP2D6 activity observed during human pregnancy could be replicated in the mouse, and explore possible mechanisms of increased CYP2D6 activity during pregnancy. Cyp2d11, Cyp2d22, Cyp2d26 and Cyp2d40 mRNA was increased (P < 0.05) on gestational days (GD) 15 and 19 compared with the non-pregnant controls. There was no change (P > 0.05) in Cyp2d9 and Cyp2d10 mRNA. In agreement with the increased Cyp2d mRNA, Cyp2d-mediated dextrorphan formation from dextromethorphan was increased 2.7-fold (P < 0.05) on GD19 (56.8±39.4 pmol/min/mg protein) when compared with the non-pregnant controls (20.8±11.2 pmol/min/mg protein). An increase in Cyp26a1 mRNA (10-fold) and retinoic acid receptor (Rar)β mRNA (2.8-fold) was also observed during pregnancy. The increase in Cyp26a1 and Rarβ mRNA during pregnancy indicates increased retinoic acid signaling in the liver during pregnancy. A putative retinoic acid response element was identified within the Cyp2d40 promoter and the mRNA of Cyp2d40 correlated (P < 0.05) with Cyp26a1 and Rarβ. These results show that Cyp2d mRNA is increased during mouse pregnancy the and mouse may provide a suitable model to investigate the mechanisms underlying the increased clearance of CYP2D6 probes observed during human pregnancy. Our findings also suggest that retinoic acid signaling in the liver is increased during pregnancy, which may have broader implications to energy homeostasis in the liver during pregnancy.

Retinoic Acid Enhances TRAIL-Induced Apoptosis in Cancer Cells by Upregulating TRAIL Receptor 1 Expression

Many human cancer cells are sensitive to killing by the proapoptotic ligand TNF-related apoptosis-inducing ligand (TRAIL), which is under study for cancer treatment in clinical trials. The TRAIL receptor (TRAIL-R1; also known as death receptor 4) is a transmembrane receptor that mediates TRAIL-induced apoptosis in cancer cells. In this study, we show that retinoids sensitize cancer cells to TRAIL-induced apoptosis by upregulating expression of TRAIL-R1. All-trans retinoic acid (ATRA) upregulated TRAIL-R1 expression in human cancer cells at the transcriptional level. The ability of ATRA to activate TRAIL-R1 expression was inhibited by retinoic acid receptor (RAR) antagonists or siRNAs, but augmented by several RAR agonists. In analyzing how ATRA induces RAR-dependent transcriptional upregulation of TRAIL-R1, we identified 2 putative retinoic acid response elements termed Pal-17 (a palindrome separated by 17 bases) and DR-11 (a direct repeat separated by 11 bases) in the 5'-flanking region of TRAIL-R1 gene. Deletion of DR-11, but not Pal-17, abrogated the ability of ATRA to stimulate TRAIL-R1 promoter activity. Consistent with this observation, RAR binding to DR-11, but not to Pal-17, was detected by chromatin immunoprecipitation assay in ATRA-treated cells, arguing that DR-11 was responsible for ATRA-mediated activation of the TRAIL-R1 gene. ATRA augmented TRAIL-induced apoptosis of cancer cells, and this activity was attenuated by a blockade to upregulation of TRAIL-R1 expression. Taken together, our findings establish that ATRA accentuates TRAIL-induced apoptosis, reveal a novel mechanism by which retinoids modulate apoptosis, and suggest a novel strategy to augment the anti-cancer activity of TRAIL.

Epidermal expression of Hox1 is directly activated by retinoic acid in the Ciona intestinalis embryo

Hox genes play important roles in the specification of spatial identity during development of vertebrate embryos. Retinoic acid regulates the transcription of Hox genes in vertebrates. We identified an epidermal enhancer in the 5' flanking region of an ortholog of Hox1 (Ci-Hox1) in the ascidian Ciona intestinalis. This enhancer element drives the transcription of a lacZ reporter gene in the epidermis in the posterior trunk and the anterior tail region of tailbud-stage embryos. Inhibition of retinoic acid synthesis resulted in inactivation of the expression of the reporter gene. The enhancer contains a putative retinoic acid response element. When this element was mutagenized, the expression of the reporter gene disappeared from the epidermis. This sequence was also required for the response to exogenously administered retinoic acid. A heterodimeric nuclear receptor, consisting of the retinoic acid receptor and retinoid X receptor, bound to this sequence. These results indicate that retinoic acid directly activates the epidermal enhancer of Ci-Hox1. This is the first demonstration that retinoic acid is necessary for endogenous gene expression in ascidian embryos.

Astrocyte Differentiation of Neural Precursor Cells is Enhanced by Retinoic Acid Through a Change in Epigenetic Modification

Neurons, astrocytes, and oligodendrocytes-the three major cell types that comprise the central nervous system-are generated from common multipotent neural precursor cells (NPCs). Members of the interleukin-6 family of cytokines, including leukemia inhibitory factor (LIF), induce astrocyte differentiation of NPCs by activating the transcription factor signal transducer and activator of transcription 3 (STAT3). We show here that retinoic acid (RA) facilitates LIF-induced astrocyte differentiation of NPCs. RA and LIF synergistically activate the promoter of gfap, which encodes the astrocytic marker glial fibrillary acidic protein, and a putative RA response element in the promoter was found to be critical for this activation. Histone H3 acetylation around the STAT-binding site in the gfap promoter was increased in NPCs treated with RA, allowing STAT3 to gain access to the promoter more efficiently. These results suggest that RA acts in concert with LIF to induce astrocyte differentiation of NPCs through an epigenetic mechanism that involves cross-talk between distinct signaling pathways.

Differential regulation of ParaHox genes by retinoic acid in the invertebrate chordate amphioxus (Branchiostoma floridae)

The ParaHox cluster is the evolutionary sister to the Hox cluster. Like the Hox cluster, the ParaHox cluster displays spatial and temporal regulation of the component genes along the anterior/posterior axis in a manner that correlates with the gene positions within the cluster (a feature called collinearity). The ParaHox cluster is however a simpler system to study because it is composed of only three genes. We provide a detailed analysis of the amphioxus ParaHox cluster and, for the first time in a single species, examine the regulation of the cluster in response to a single developmental signalling molecule, retinoic acid (RA). Embryos treated with either RA or RA antagonist display altered ParaHox gene expression: AmphiGsx expression shifts in the neural tube, and the endodermal boundary between AmphiXlox and AmphiCdx shifts its anterior/posterior position. We identified several putative retinoic acid response elements and in vitro assays suggest some may participate in RA regulation of the ParaHox genes. By comparison to vertebrate ParaHox gene regulation we explore the evolutionary implications. This work highlights how insights into the regulation and evolution of more complex vertebrate arrangements can be obtained through studies of a simpler, unduplicated amphioxus gene cluster.

The Expression Signature of M3 AML Suggests Direct and Indirect Effects of PML-RARA on Target Genes.

In order to better understand the pathogenesis of acute promyelocytic leukemia (APL, FAB M3), we sought to determine its gene expression signature by comparing the expression profiles of 14 APL samples to that of other AML subtypes (M0, M1, M2, M4, n=62) and to fractionated normal whole bone marrow cells (CD34 cells, promyelocytes, PMNs, n=5 each). We used ANOVA and SAM (Significance Analysis of Microarrays) to select genes that were highly expressed in APL cells and that displayed low to no expression in other AML subtypes. The APL signature was then further refined by filtering genes whose expression in APL was not significantly different from that of normal promyelocytes, yielding 1121 annotated genes that reliably distinguish APL from the other FAB subtypes using unsupervised hierarchical clustering, both in training and validation datasets. Fold change differences in expression between M3 and other AML FAB classes were striking, for example: GABRE 35.4, HGF 21.3, ANXA8 21.3, PTPRG 16.9, PTGDS 12.1, PPARG 11.1, STAB1 9.8. A large proportion of the APL versus other FAB dysregulome was recapitulated when we compared APL expression to that of the normal pattern of myeloid development. We identified 733 annotated genes with significantly different expression in APL versus normal myeloid cell fractions. These dysregulated genes were assigned to 4 classes:1.persistently expressed CD34 cell-specific genes,2.repressed promyelocyte-specific genes,3.prematurely expressed neutrophil-specific genes and4.genes with high expression in APL and low/no expression in normal myeloid cell fractions.Expression differences in several of the most dysregulated genes were validated by qRT-PCR. We then examined the expression of the APL signature genes in myeloid cell lines and tumors from a murine APL model. The bona fide M3 signature was not apparent in resting NB4 cells (which contain t(15;17), and which express PML-RARA), nor in PR-9 cells following Zn induction of PML-RARA expression, suggesting that neither cell line accurately models the gene expression signature of primary APL cells. Most of the nodal genes of the mCG-PML-RARA murine APL dysregulome (Yuan, et al, 2007) are similarly dysregulated in human M3 cells; however, the human and mouse dysregulomes do not completely coincide. Finally, we have begun investigating which APL signature genes are direct transcriptional targets of PML-RARA. The promoters of the APL signature genes were analyzed for the presence of known PML-RARA binding sites using multiple computational methods. The analyses demonstrated that several transcription factors (EBF3, TWIST1, SIX3, PPARG) have putative retinoic acid response elements (RAREs) in their upstream regulatory regions. Additionally, we examined the promoters of some of the most upregulated genes (HGF, PTGDS, STAB1) for known consensus sites of these transcription factors, and found that all have putative binding sites for at least one. These results suggest that PML-RARA may initiate a transcriptional cascade that relies not only on its own activity, but also on the actions of downstream transcription factors. In summary, our studies indicate that primary APL cells have a gene expression signature that is consistent and highly reproducible, but different from commonly used human APL cell lines and a mouse model of APL. The molecular mechanisms that govern this unique signature are currently under investigation.

LGL1, a novel branching morphogen in developing kidney, is induced by retinoic acid

Late-gestation lung protein 1 (LGL1) is a glycoprotein secreted by fetal lung mesenchyme that stimulates branching morphogenesis of the developing lung bud. We show that Lgl1 mRNA and protein are also expressed in mesenchymally derived lineages of fetal kidney. Although Lgl1 expression is stimulated by glucocorticoids in kidney cells, cortisol (10(-7) M) actually suppresses ureteric bud branching of fetal kidneys from HoxB7/GFP mice in explant culture. However, early branching morphogenesis in the lung and kidney is stimulated by retinoic acid, and we identified putative retinoic acid response elements in the Lgl1 promoter. All-trans-retinoic acid (10(-6) M) stimulated Lgl1 promoter activity and endogenous Lgl1 mRNA expression in vitro. Branching of cultured fetal kidney explants was increased in the presence of all-trans retinoic acid (10(-6) M). Heterozygous Lgl1 knockout mice were crossed to HoxB7/GFP mice to visualize the extent of ureteric bud branching at fetal stages. At embryonic (E) days E12.5-E13.0, mutant Lgl1(+/-) embryos showed a 20% reduction in ureteric bud branching compared with wild-type littermates. We propose a model in which retinoic acid stimulates branching morphogenesis by activating Lgl1 early in development. The prominent effects of glucocorticoids on Lgl1 expression in late lung development suggest a second role for LGL1 in alveolar maturation.

Crk-associated substrate (Cas) family member, NEDD9, is regulated in human neuroblastoma cells and in the embryonic hindbrain by all-trans retinoic acid.

The vitamin A metabolite, all-trans retinoic acid (atRA), plays an essential role in vertebrate embryogenesis, including development of the nervous system. In the human neuroblastoma cell line, SH-SY5Y, atRA rapidly induces (within 4 hr) the expression of the Crk-associated substrate (Cas) family member, neural precursor cell-expressed, developmentally down-regulated gene 9 (NEDD9) also called the human enhancer of filamentation (HEF1). NEDD9 is expressed in the developing hindbrain (5-somite stage) in the presumptive rhombomeres 2, 3, and 5 before the onset of overt segmentation. Exposure of rat embryos to excess atRA at times ranging from E9.25 to E12 leads to altered NEDD9 expression in the developing hindbrain within 6 hr. NEDD9 expression is also perturbed in vitamin A-deficient embryos. A putative retinoic acid response element in the 5' region of the NEDD9 promoter binds specifically to a RXR/RAR heterodimer and forms a higher molecular weight complex upon addition of a retinoic acid receptor-specific antibody. Regulation of NEDD9 may be an important means whereby atRA promotes cell spreading and neurite outgrowth in SH-SY5Y human neuroblastoma cells, and NEDD9 represents a new downstream target of atRA and its receptors in the developing hindbrain.

Retinoids induce the PAI-1 gene expression through tyrosine kinase-dependent pathways in vascular smooth muscle cells.

Retinoids exert their pleiotropic effects on several pathophysiologic processes, including neointima formation after experimental vascular injury. Plasminogen activator inhibitor-1 (PAI-1) has been proposed to play an inhibitory role in arterial neointima formation after injury. We examined whether retinoids regulate PAI-1 expression in cultured vascular smooth muscle cells (SMCs). Northern blot analysis showed that all-trans retinoic acid (atRA) and 9-cis retinoic acid (9cRA) increased PAI-1 mRNA levels in a dose-dependent manner. These responses were completely inhibited by tyrosine kinase inhibitors. The half-life of PAI-1 was not affected by atRA, suggesting that induction of PAI-1 mRNA was mainly regulated at the transcriptional levels. Stable and transient transfection assays of the human PAI-1 promoter-luciferase constructs indicate that DNA sequence responsive to either ligand-stimulated or overexpressed retinoic acid receptor-alpha expression vector lies downstream of -363 relative to the transcription start site, where no putative retinoic acid response element is found. These results indicate that atRA and 9cRA increase PAI-1 gene transcription through pathways involving tyrosine kinases in SMCs. Because PAI-1 inhibits the production of fibrinolytic protein plasmin that facilitates SMC migration, induction of the PAI-1 gene expression by atRA may at least partly account for the role of atRA as an important inhibitor of neointima formation.

Retinoic Acid Induces Gpx2 Gene Expression in MCF-7 Human Breast Cancer Cells

We showed previously that the selenium-dependent glutathione peroxidase, GPX-GI, encoded by the Gpx2 gene, is highly expressed in the epithelium of the gastrointestinal (GI) tract and sporadically in breast tissue. To investigate whether Gpx2 gene expression is epithelium specific, we used in situ hybridization to show that Gpx2 mRNA is highly expressed in the crypt epithelium of human intestine. We also used Northern analysis to study human breast cells and found Gpx2 mRNA in human mammary epithelial cell lines as well as freshly isolated normal breast epithelial cells. Because we identified three putative retinoic acid response elements (RARE) in the Gpx2 gene, we examined the regulation of the Gpx2 gene expression by all-trans retinoic acid (RA) in RA-sensitive MCF-7 cells and RA-resistant HT29 cells. Without RA, MCF-7 cells had very low levels of Gpx2 mRNA and a low level of glutathione peroxidase (GPX) activity (17 mU/mg protein), whereas HT29 cells had a high level of Gpx2 mRNA and GPX activity (200 mU/mg protein). RA treatment increased Gpx2 mRNA level 3- to 11-fold and resulted in a fourfold increase of GPX activity (80 mU/mg protein) in MCF-7 cells. Neither Gpx2 mRNA level nor GPX activity was increased in HT29 cells. These results show that the Gpx2 gene is expressed in both breast and intestinal epithelium cells, and suggest that its expression can be highly regulated by retinoic acid, a known differentiation agent.

NEGATIVE REGULATION OF THE INTERLEUKIN (IL)-2 RECEPTOR α CHAIN (CD25) PROMOTER REGION IN HUMAN B LYMPHOCYTES

Studies of the CD25 gene promoter region in T cells have revealed the presence of a 31-bp region, including an 11-bp negative regulatory element (NRE), which profoundly suppresses CD25 expression. This report illustrates that the same region acts as a negative regulator of CD25 expression in human B lymphocytes. Human B cells contain DNA-binding protein activities which bind specifically to an oligonucleotide equivalent to the 11-bp core region of the NRE, and stimulation of tonsillar B cells or Daudi Burkitt's lymphoma B cells with interleukin 4 (IL-4) results in loss of binding activity for oligonucleotides containing the NRE; in contrast, IL-4 enhanced binding activity for the NRE in the Jurkat T cell line. Transient transfection analyses using deletion mutants lacking both the 11-bp core NRE and both the NRE and an adjacent putative retinoic acid response element (RARE) motif illustrated that the NRE element is a functional suppressor of CD25 transcription in B cells. Thus, deletion of the NRE element increased the basal level of CD25 promoter activity and also conferred IL-4 inducibility on reporter gene expression in transiently transfected tonsillar B lymphocytes.

Essential cis-Acting Elements in Rat Uncoupling Protein Gene Are in an Enhancer Containing a Complex Retinoic Acid Response Domain

Transgenic mice were generated with a transgene containing the 211-base pair (bp) enhancer and 0.4 kilobase pairs of 5'-flanking DNA of the uncoupling protein (ucp) gene. Expression of this transgene was restricted to brown adipose tissue and was inducible by cold exposure or treatment of transgenic mice by norepinephrine, retinoic acid (RA), or CL-316,243 beta3-adrenoreceptor agonist. A search for retinoic acid response elements in the ucp gene enhancer was undertaken using mutagenesis and transfection of cultured cells with chloramphenicol acetyltransferase constructs. Deletion or mutations of several putative retinoic acid response elements were ineffective. Mutations of a TGAATCA region dramatically decreased the transcriptional activity in the presence of RA. In vitro this region was able to bind a complex containing proteins recognized by antibodies against Jun or Fos. Mutations of an adjacent region related to an inverted repeat of type 2 also markedly decreased RA effect. This region was able to bind in vitro retinoid X receptor alpha and retinoic acid receptor beta. The two regions form an activating region between bp -2421 and -2402 (referred to as the ucp gene-activating region), which has an enhancer activity but cannot confer RA response to a promoter. This response was obtained with a larger DNA fragment (bp -2489 to -2398) constituting a complex RA response domain.

All-trans- and 9-cis-retinoic acid enhance the cholinergic properties of a murine septal cell line: evidence that the effects are mediated by activation of retinoic acid receptor-alpha.

We investigated the effects of retinoids on the cholinergic properties of a murine septal cell line, SN56. Treatment of the cells with all-trans-retinol (vitamin A), all-trans-retinal, all-trans-retinoic acid (t-RA), 9-cis-retinoic acid (9c-RA), or 13-cis-retinoic acid caused time- and concentration-dependent increases in choline acetyltransferase activity (up to 3.4-fold) and in intracellular acetylcholine levels (up to 2.5-fold, with respective EC50 values of 68, 50, 18, 15, and 56 nM). Furthermore, treatment with either t-RA or 9c-RA at 1 microM for 48 h resulted in an increase in the expression of choline acetyltransferase mRNA by threefold that of controls. These data and the presence of putative retinoic acid response elements in the 5' region of the murine choline acetyltransferase gene indicate that retinoids stimulate choline acetyltransferase transcription in murine cholinergic neurons. No additivity or synergism was observed between the effects of t-RA and 9c-RA on any of these cholinergic properties of SN56 cells, suggesting a common mechanism of action of the two retinoids. However, a combined treatment with t-RA and forskolin, which activates adenylate cyclase, resulted in an additive increase in acetylcholine content. Using an antagonist selective for the retinoic acid receptor-alpha subtype, Ro 41-5253, we found that the effects of t-RA and 9c-RA on acetylcholine levels were abolished. An agonist selective for retinoic acid receptor-alpha, Ro 40-6055, increased acetylcholine levels to a similar extent as t-RA and 9c-RA, and this effect was blocked by the antagonist.(ABSTRACT TRUNCATED AT 250 WORDS)

Retinoid X receptor RXR alpha binds to and trans-activates the hepatitis B virus enhancer.

A retinoid X receptor (RXR) response element was located within the functionally defined hepatitis B virus (HBV) enhancer element. A short segment of the enhancer that contains this region has been shown with genetic analysis to play a key role in the regulation of enhancer function and to represent a major determinant of liver-specific activity. Both the full-length protein and the DNA-binding domain of the liver-specific receptor RXR alpha bound to the putative retinoic acid response element in the HBV enhancer. In vivo, an HBV enhancer-reporter gene construct responds to induction with retinoic acid when cotransfected with an RXR alpha expression vector. A single-base transition (G----A) in the HBV retinoic acid response element leads to a dramatic reduction both in the in vitro binding activity of RXR alpha and the in vivo activity of the HBV enhancer. Thus, retinoic acid and the RXR alpha are implicated as being significant determinants in the liver-specific regulation of HBV gene expression and the resultant disease pathogenesis.